Combined Oxygen and Nitrous Oxide Microsensor for Denitrification Studies

The construction of a microsensor which can be used to measure O₂ and N₂O simultaneously is described. The microsensor exhibited a linear response to both O₂ and N₂O, and the response to N₂O was independent of the O₂ concentration and vice versa. The N₂O detection limit of a microsensor with a tip diameter of 20 μm was around 1 μmol liter⁻¹. The signals for O₂ and N₂O were affected by hydrogen sulfide, but other interfering agents were not observed in the biofilms and sediments analyzed. Microprofiles of O₂ and N₂O were measured in a biofilm which was exposed to acetylene to block the N₂O reductase activity of denitrifying bacteria. O₂ penetrated about 0.5 mm into the biofilm and was not affected by acetylene, but the N₂O concentration at 1.4 mm depth increased from 32 to 411 μmol liter⁻¹ after the addition of the inhibitor. The shape of the N₂O profile after the addition of acetylene showed that denitrification (denitrifying activity) was detectable in all anoxic layers of the biofilm.     

Linear dichroism and orientational studies of carotenoid Langmuir-Blodgett films

The linear dichroism of single monolayers of lutein, zeaxanthin and a mixture of lutein and synthetic phosphatidylcholine has been measured. The angle of orientation of the carotenoid molecules was found to lie between 45° and 51° relative to the plane of the solid support. Although the adsorbed monolayers were mostly in a monomeric state, microscopic observations, as well as the II-A isotherms, indicated the existence of crystalline islets. The results have been interpreted in connection with Haidinger's polarization brushes.     

Stereoselective effects in the interactions of pterin cofactors with rat-liver phenylalanine 4-monooxygenase

The (6R) and (6S) epimers of l-erythro-tetrahydrobiopterin (BH4) and some of its structural analogs, were tested as cofactors and non-covalent effectors in the phenylalanine 4-monooxygenase (phenylalanine hydroxylase, EC 1.14.16.1) reaction. The oxidation-reduction potentials (Em,7) of the free (not enzyme-bound) form of the (6R) and (6S) epimers were rather similar (range 174-184 mV) for the oxidation of tetrahydropterins to quinonoid dihydropterins. Rapid-mixing kinetic experiments were performed at 20 degrees C under conditions which allow only a few turnover reactions of the enzyme. Three main oxidation products were identified spectroscopically at pH 6.8 for all three tetrahydropterins tested: the C(4a)-hydroxy derivatives, the quinonoid dihydropterins, and the stable 7,8-dihydropterins (in that sequence). The formation of the C(4a)-hydroxy forms closely paralleled that of tyrosine, and supports the proposal that this covalent adduct is formed as an immediate product on completion of the catalytic cycle. Assay of the initial rate of C(4a)-hydroxy derivative formation represents a new approach in kinetic studies of this enzyme, and the kinetic parameters obtained for the phenylalanine-activated enzyme are presented. The affinity of binding of (6R)-BH4 and (6S)-BH4 to phenylalanine hydroxylase was also estimated on the basis of their quenching of the intrinsic tryptophan fluorescence of the enzyme. The apparent affinities were found to correspond well to the Km values estimated in kinetic studies of the hydroxylation reaction with the phenylalanine activated enzyme, i.e. higher for (6R)-BH4 than for (6S)-BH4. The lower V value observed for the native enzyme with the (6R) epimer in steady-state kinetics is explained by its higher potency as a negative effector, since the oxidation-reduction potentials of the two diastereomers were similar. Dihydrobiopterin (BH2) was found to inhibit the hydroxylation reaction and quenched the intrinsic tryptophan fluorescence of the enzyme with the same concentration dependence as that observed with (6S)-BH4.     

A burst of CO2 from photosynthesizing leaves after a temperature decrease under constant light conditions

A transient CO₂ burst from seedlings of some plant species was observed after a rapid temperature decrease. The magnitude of the CO₂ release depended on initial temperature, oxygen concentration and light intensity. To obtain a maximal value of CO₂ release, the temperature had to decrease by more than 8°C. The phenomenon was detected only in the light, and was confined to C₃ species. It was inhibited by low oxygen concentration, indicating its possible connection with photorespiration.     

Sodium arsenite enhances the cytotoxicity, clastogenicity, and 6-thioguanine-resistant mutagenicity of ultraviolet light in Chinese hamster ovary cells

Cytotoxicity, chromosome aberrations, and mutations to 6-thioguanine resistance were synergistically increased by incubating the ultraviolet light (UV)-irradiated Chinese hamster ovary (CHO) cells in medium containing sodium arsenite. However, the frequencies of sister-chromatid exchanges and mutations to ouabain resistance induced by UV were not synergistically increased by sodium arsenite. The synergistic effect of sodium arsenite on UV-induced chromosome aberrations varied with cell-harvesting time and decreased with increasing time intervals between UV and sodium arsenite treatments.     

Requirement for the action of endogenous ethylene during germination of non-dormant seeds of Amaranthus caudatus

The role of endogenous ethylene during germination of non-dormant seeds of Amaranthus caudatus L. was investigated. The seeds readily germinated in water and darkness at 24°C. Application of ethylene or of its precursor I-aminocyclopropane-I-carboxylic acid (ACC) slightly increased the rate of germination. Both compounds effectively antagonized osmotic inhibition by polyethyleneglycol. Application of aminoethoxyvinylglycine (AVG) reduced ethylene production by 90% but did not inhibit germination. However, germination was inhibited by 2,5-norbornadiene, a competitive inhibitor of ethylene action. This inhibition was counteracted by ethylene, ethephon or ACC and enforced by AVG. It is concluded that the action of endogenous ethylene is an indispensable factor during germination of non-dormant seeds of A. caudatus. Ethylene action is required from the start of imbibition on. In water, low levels of endogenous ethylene are sufficient for this action. PEG increased the ethylene requirement considerably.     

UV-microscopic studies on the vacuolar changes caused by the flavone “aglycone” isovitexin inSilene pratensis plants

Summary UV-microscopic and chromatographic studies have been performed on the variation in contents and configuration of the flavones present in epidermal cells of the petals, stem leaves, rosette leaves and cotyledons ofSilene pratensis plants. Most of the flavone contents is located in the vacuole of the upper epidermis cells, the concentration depending on the light intensity at which the plants were grown. In plants able to glycosylate isovitexin in the petals (genotypegG/. gl/gl fg/fg, accumulating isovitexin 7-O-glucoside) the vacuole is completely filled with the UV absorbing flavone. In plants which are unable to glycosylate isovitexin in their petals (genotypeg/g gl/gl fg/fg, accumulating only isovitexin) the upper epidermal cells of stem leaves and petals contain droplet like structures in their vacuoles. At high light intensities these structures increase in mass and become detectable in the visible light. These denser structures often condense to structures with radiating threads.As compared with the accumulation of isovitexin in upper epidermal cells of stem leaves and petals in genotypeg/g gl/gl fg/fg, the cotyledons and the rosette leaves contain two isovitexin glycosides. In the latter organs the upper epidermal cells are very similar to the upper epidermal cells fromgG/. gl/gl fg/fg plants, having a vacuole filled with UV absorbing material. It appears therefore that isovitexin itself causes the formation of the structurés in the cells. It was shown by varying the light intensity that a relative high concentration of isovitexin is necessary for the droplet like structures to appear. Still higher concentrations are needed for the formation of the structures with radiating threads. It is hypothesized that isovitexin interferes with the energy supply of the cells, which therefore are not able to maintain their turgor.     

A high-affinity folate binding protein in normal human leukocytes: Ligand binding characteristics,ionic charge and molecular size

High-affinity binding of [³H]folate to supernatant from homogenized human leukocytes containing large amounts of binding protein displayed apparent positive cooperativity. The DEAE-Sepharose^® CL-6B chromatographic profile of the supernatant at pH 6.3 contained a major peak of folate binding (M_r approx. 25 000) in the front effluent and a smaller more acidic peak (M_r approx. 25 000) that emerged after a rise in NaCl from 30 mmol/l to 1 mol/l. Triton X-100 solubilized ceil sediment from the leukocyte homogenate contained some high-affinity folate binding activity (M_r approx 25 000), typically 5–10% of the total binding activity.